Title: Deciphering tumour heterogeneity and transcriptomic profiles of circulating tumor cells in archived metastatic colorectal cancer samples via single-cell mRNA sequencing
Abstract:
Molecular single-cell analyses have revolutionised the current understanding of complex biological processes by revealing rare cell populations, including circulating tumour cells (CTCs), that were previously masked by bulk analyses. While CTCs have proven to be valuable prognostic markers for metastatic colorectal cancer (mCRC), their characterisation at the singe cell level remains limited.
Purpose: This study aimed to perform an in-depth characterisation of CTCs in mCRC via single-cell RNA-sequencing (scRNA-seq).
Methods: Thirteen out of 60 CRC patients were identified via a CTC biomarker panel - the MILLIPLEX® MAP kit-human circulating cancer biomarker magnetic bead panel 4. Pure CTC suspension from pooled PBMC samples was then isolated via an antibody-free negative enrichment using the EasySep human CD45 depletion kit II. Single CTCs were sorted and barcoded using BD Rhapsody single-cell analysis system, followed by whole transcriptomics amplification and NextSeq sequencing. Bioinformatic analyses were performed using Seven Bridges, ICARUS and GeneCodis4 online analysis tools.
Results: Three potential serum markers for CTC screening (L1CAM, CA9, HPN) in mCRC were identified, and 507 single CTCs with a 0.9% cell multiplet rate were captured. Six distinct CTC clusters were identified, and differential expression analyses highlighted the significant overexpression of the MALAT1 gene across multiple clusters. A comparison between clusters 4 and 1 revealed four significant differentially expressed genes including SLC4A1 and BCL2L1, associated with CRC proliferation, cell cycle and apoptosis. Gene ontology and pathway analyses predicted the interference of PABPC1 in the nonsense-mediated decay pathway, potentially inhibiting MALAT1 degradation through NF-κB and TGF-β signalling pathways mediated by UPF1. Co-expression of PABPC1 and MALAT1 shows promise as a potential biomarker signature for mCRC CTCs. Additionally, CD74 was found to contribute to the survival of tumour cells by evading immune attacks through immunoediting surface antigen presentation.
Conclusion: scRNA-seq identified unique genes associated with CRC metastasis, highlighting the presence of tumour heterogeneity within CTCs from mCRC. These findings have significant implications for the development of liquid biopsy-based methods in identifying biomarkers for mCRC and guiding personalised treatment approaches in the future
Audience Take Away Notes:
- Cancer cells from blood samples of patients with late-stage colon cancer, contain important information that can help personalise cancer treatment.
- This research demonstrates that archived circulating tumour cells (CTCs), even those stored for over five years, can be effectively used for single-cell RNA sequencing. This finding is valuable for other researchers as it simplifies the sample collection process, allowing them to utilise existing archived samples for single-cell analysis. By showing the feasibility of using long-term stored samples, this study provides a cost-effective and efficient approach for future research in the field.
- The identification of potential serum markers (L1CAM, CA9, HPN) for CTC screening in late-stage colon cancer, provide insights into the development of non-invasive diagnostic approaches.
- The discovery of distinct CTC clusters and differential gene expression patterns related to cancer proliferation, metastasis, and drug resistance, highlights the existence of tumour heterogeneity
